Picornaviruses are major human pathogens, and their worldwide prevalence is responsible for some of the highest morbidities of all known viruses. Due to the limited coding capacity of picornavirus genomic RNAs, host proteins play critical roles during viral translation and RNA replication in the cytoplasm of infected cells. A number of these proteins are normally localized to the nucleus of uninfected human cells. The experiments proposed in this application aim to define the changing protein landscape in human rhinovirus infected cells that is brought about by viral-induced modifications of the nucleo-cytoplasmic trafficking machinery of the host cell. The proposal will explore the unique mechanisms that rhinovirus has evolved to take advantage of host nuclear proteins which play key roles in IRES-mediated translation and viral RNA synthesis during its intracellular replication cycle. By interacting with normally nuclear-resident proteins during its cytoplasmic replication cycle, rhinovirus expands the repertoire of host cell functions it can hijack for its own purposes. Using an unbiased proteomics approach, this application will address the hypothesis that human rhinovirus induces a re-partitioning of host cell proteins between the nucleus and the cytoplasm to potentiate steps in its replication cycle. The proposed experiments will expand upon a recent discovery that rhinovirus infection of human lung fibroblast cells results in a different set of virus-host interactions compared to what is observed during a rhinovirus or poliovirus infection of HeLa cells. Two experimental aims will be carried out to discover if human rhinovirus uses a distinct set of host proteins and mechanisms during replication in lung cells aided, in part, by the novel viral-induced redistribution of resident nuclear proteins to the cytoplasm of infected cells: (1) Generate a nuclear versus cytoplasmic human cell proteome during a human rhinovirus infection, and (2) Identify candidate host proteins and determine their functional significance for human rhinovirus replication. Overall, determining how rhinoviruses and other picornaviruses take advantage of altered nucleo-cytoplasmic trafficking should reveal new mechanistic insights into RNA virus replication and identify novel targets for antiviral therapeutics at the virus-host interface.